Traumatic injury is a major, largely unrecognized public health problem in North America that cuts across age, race, gender, and economic boundaries. The resulting loss of productive life years exceeds that of any other diseases in the US, with societal costs of over $260 billion each year. Most trauma deaths result from insufficient tissue perfusion due to excessive blood loss or from the development of inflammation, infection, and organ damage after seemingly adequate resuscitation. Clinical management of hemorrhagic shock relies on massive and rapid infusion of fluids to maintain blood pressure. However, the majority of victims with severe blood loss do not respond well to fluid restoration. Development of effective strategies for resuscitation of traumatic blood loss, therefore, is urgently needed. In this proposal, we will continue our discovery of a novel proinflammatory mediator/cytokine, cold-inducible RNA-binding protein (CIRP). We have shown that CIRP expression is upregulated in an animal model of hemorrhage and in surgical ICU patients. By using newly-expressed and purified recombinant murine CIRP (rmCIRP), we have demonstrated that administration of rmCIRP in healthy animals induces acute inflammation and causes tissue injury. In addition, anti-rmCIRP antibodies attenuate hemorrhage-induced proinflammatory cytokines, neutrophil accumulation, and tissue injury. Anti-rmCIRP antibodies also improve the survival rate from 43% to 85% after hemorrhage and fluid resuscitation. Moreover, hemorrhage-induced mortality is much lower in CIRP knockout mice (CIRP-/-) than that of wild-type mice. By using GFP-CIRP expression plasmid, we were able to show CIRP translocation from the nucleus to cytoplasm under hypoxic conditions. We have determined that TLR4, rather than TLR2 or RAGE, serves as CIRP receptor. Biacore(R) biosensor assay showed a high binding affinity (Kd =1.55M) between human CIRP and human TLR4/MD2 complex. Based on these novel findings, we hypothesize that CIRP plays an important role in hemorrhage-induced tissue injury and mortality, and that CIRP antagonists should provide a novel adjunct therapy for traumatic hemorrhage resuscitation. Accordingly, three specific aims are proposed: (1) to study the mechanism responsible for hemorrhage-induced upregulation of CIRP; (2) to define the mechanism by which CIRP produces inflammation and tissue injury; and (3) to determine whether delayed CIRP inhibition after hemorrhage with crystalloid versus blood resuscitation is also beneficial. These proposed studies should open up a new research field for the development of innovative therapeutic agents as resuscitation adjuncts for victims with traumatic hemorrhage.